The development of synthetic polynucleotide delivery systems is hampered by the extreme instability of these colloidal suspensions as liquid formulations. This instability also affects basic research by forcing scientists to conduct experiments with vector preparationsthat are prone to significant batch-to-batch variability. As a result, there is interest in developing methods that would allow synthetic vectors to be formulated as stable, dehydrated preparations that could be prepared in large batches, and shipped and stored at ambient temperatures. The work proposed here focuses on developing methods of dehydrating synthetic vectors such that their physical characteristics and biological activity are maintained during both acute lyophilization stress and prolonged storage. Previous work on these problems has demonstratedthat stabilization can be achieved during acute lyophilization stress by employing high amounts of sugars. Unfortunately, the amount of sugar needed for stabilization is not osmotically compatible with intramuscular or subcutaneous injection;the preferred method of administration for many applications, e.g., DNAvaccines. We propose mechanistic studies to determine the causative effects of vector aggregation (the major mechanism of damage) during acute lyophilization stress, and pursue strategies that achieve stability at isotonic osmolalities. The findings from these initial studies will be applied to experiments investigating two approaches to enhance storage stability. In the first approach, formulations will be directly assessed for their ability to reduce the accumulation of reactive oxygen species during storage. These experiments utilize a novel fluorescence technique that, for the first time, allows the generation of oxygen radicals in dried preparations to be monitored. The second approach is consistent with recent reports and questions the conventional dogma in the field of solid-state stability that "drier is better". Ultimately, the results from these approaches will be combined in a 2-year storage stability study on fully-optimized formulations. Recognizing that different types of synthetic vectors are being optimized for gene/polynucleotide delivery, these experiments employ different model vectors (e.g., lipoplexes and polyplexes) in order to develop rational formulation guidelines that are generally applicable to the stabilization of macromolecular complexes (e.g., vaccines, viruses, nanoparticles) during freeze-drying and storage.